ADAMTS-7 modulates atherosclerotic plaque formation by degradation of TIMP-1

Background The ADAMTS7 locus was genome-wide significantly associated with coronary artery disease (CAD). Lack of the extracellular matrix (ECM) protease ADAMTS-7 was shown to reduce atherosclerotic plaque formation. Here, we sought to identify molecular mechanisms and downstream targets of ADAMTS-7 mediating risk of atherosclerosis. Methods Targets of ADAMTS-7 were identified by high-resolution mass spectrometry of atherosclerotic plaques from Apoe-/- and Apoe-/-Adamts7-/- mice. ECM proteins were identified using solubility profiling. Putative targets were validated using immunofluorescence, in vitro degradation assays, co-immunoprecipitation, and Förster resonance energy transfer (FRET)-based protein-protein interaction assays. ADAMTS7 expression was measured in fibrous caps of human carotid artery plaques. Results In humans, ADAMTS7 expression was higher in caps of unstable as compared to stable carotid plaques. Compared to Apoe-/- mice, atherosclerotic aortas of Apoe-/- mice lacking Adamts-7 (Apoe-/-Adamts7-/-) contained higher protein levels of tissue inhibitor of metalloproteases 1 (Timp-1). In co-immunoprecipitation experiments, the catalytic domain of ADAMTS-7 bound to TIMP-1, which was degraded in the presence of ADAMTS-7 in vitro. ADAMTS-7 reduced the inhibitory capacity of TIMP-1 at its canonical target matrix metalloprotease 9 (MMP-9). As a downstream mechanism, we investigated collagen content in plaques of Apoe-/- and Apoe-/-Adamts7-/- mice after Western diet. Picrosirius red staining of the aortic root revealed less collagen as a readout of higher MMP-9 activity in Apoe-/- as compared to Apoe-/- Adamts7-/- mice. In order to facilitate high-throughput screening for ADAMTS-7 inhibitors with the aim to decrease TIMP-1 degradation, we designed a FRET-based assay targeting the ADAMTS-7 catalytic site. Conclusions ADAMTS-7, which is induced in unstable atherosclerotic plaques, decreases TIMP-1 stability reducing its inhibitory effect on MMP-9, which is known to promote collagen degradation and is likewise associated with CAD. Disrupting the interaction of ADAMTS-7 and TIMP-1 might be a strategy to increase collagen content and plaque stability for reduction of atherosclerosis-related events.


Introduction
Atherosclerosis of the coronary arteries is promoted by several risk factors 1 , such as hypertension, diabetes, hypercholesterolemia, smoking, male gender, as well as increasing age and genetic disposition 2 .In recent years, genome-wide association studies (GWAS) have identified multiple common alleles that underlie the genetic risk 3 .One of the strongest loci associated by GWAS with coronary artery disease (CAD) and myocardial infarction (MI) risk [4][5][6] represents the extracellular matrix (ECM) protease ADAMTS-7 7 .
In experimental studies, lack of Adamts-7, the murine counterpart, was associated with beneficial vascular remodeling 8 and reduced atherosclerotic plaque formation 9 .Recently, in an experimental study vaccination against ADAMTS-7 successfully reduced atherosclerotic plaque formation under pro-atherogenic conditions and prevented neointima formation and in-stent restenosis 10 .The effects of ADAMTS-7 on vascular remodeling can be explained by degradation of the substrate cartilage oligomeric matrix protein (COMP) 11 , which was also found to mediate ADAMTS-7 related effects in rheumatoid arthritis and represents its first known substrate.Another binding partner of ADAMTS-7 is thrombospondin-1 (THBS1 or TSP1) 8 .In contrast, the precise downstream mechanisms involving ADAMTS-7 in atherosclerotic plaque formation remain incompletely understood.
Here, we observed increased ADAMTS-7 expression in unstable as compared to stable human plaques.In mice, we used a proteome-wide analysis of atherosclerotic aortas to find downstream targets of pro-atherogenic ADAMTS-7.We identified the endogenous inhibitor of metalloproteinases TIMP-1 as a novel target of ADAMTS-7 and verified their interaction at the catalytic site of ADAMTS-7 in vitro.As a functional consequence of reduced TIMP-1 levels, increased MMP-9 activity led to reduced plaque collagen content which might influence plaque stability.Finally, we explored the interaction between ADAMTS-7 and TIMP-1 to establish a screening assay for the identification of ADAMTS-7 inhibitors.

Methods
A detailed, expanded Methods section is available in the Supplemental Material.

Mouse models
Animal experiments were conducted in accordance with the German legislation on protection of animals and approved by the local animal care committee (122-4 (108-9/11)).
Mice had ad libitum access to food and water and were housed under a 12 h light-dark cycle.
Apoe-/-Adamts7-/-mice were generated by crossbreeding Adamts7-/-8 with Apoe tm1Unc (purchased from the Jackson Laboratories, Bar Harbor, USA; subsequently termed Apoe-/-) mice for more than four generations.All experiments were performed on Apoe-/-and Apoe-/-Adamts7-/-mice that were fed a Western Diet (TD88137, Harlan) for either twelve or 16 weeks (specified below) starting at the age of eight weeks for determination of plaque collagen content or high-resolution mass spectrometry of atherosclerotic plaques, respectively.
For analysis of plaque collagen content, male and female mice at a 1:1 ratio were used and sacrificed at an age of 20 weeks (twelve weeks Western Diet).Aortic roots were embedded in optimal cutting temperature compound (Sakura Finetek, Tokyo, Japan) and snap frozen to -80 °C.Frozen samples were cut into 5 μm sections and applied to microscope slides.
From the onset of aortic valves, every fifth slide was subjected to tissue staining as described below.
For high-resolution mass spectrometry analysis of atherosclerotic plaque ECM, male animals were analyzed and sacrificed at age 24 weeks (16 weeks Western Diet) by an overdose of pentobarbital (600 mg/kg; Release® 300 mg/ml, WDT, Garbsen, Germany).The arterial tree was perfused through the left ventricle with 20 ml 0.9 % sodium chloride solution.For quantitative detergent solubility profiling aortas were dissected from the ascending part until the bifurcation to the renal arteries and snap frozen in liquid nitrogen and subsequently stored at -80 °C.Sample preparation for extraction of aorta ECM, proteomic analysis and computational mass spectrometry data analysis were performed as described previously 12 .Of note, the data of the Apoe-/-control group were used as cohort #2 in our previous study 12 .

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Europe PMC Funders Author Manuscripts Inclusion and exclusion criteria: All animals which completed the studies were included in the overall analysis.Only animals that died or had to be sacrificed during the experiment were excluded.

LC-MS analysis of atherosclerotic plaques ECM
Statistical analysis of label free quantification (LFQ) derived protein expression data was performed using Perseus.Protein entries referring to potential contaminants, proteins identified by matches to the decoy reverse database, and proteins identified only by modified sites, were removed.LFQ intensity values were normalized by log2 transformation and missing values were imputed using the mixed imputation approach.With this method, we looked at missing values in samples belonging to the same group and impute with k-nearest neighbors if there was at least 60% of valid values in that group, for that protein.
The remaining missing values were imputed with the MinProb method (random draws from a Gaussian distribution; width = 0.3 and downshift = 1.8) 13 .Differentially enriched proteins were identified by unpaired t-tests with permutation-based FDR correction for multiple hypothesis (FDR <0.01, s0=1, permutations=250) following the SAM algorithm 14 .Significantly regulated proteins were colored in red and blue in the volcano plots for up-and downregulated hits, respectively.

Immunoblotting
The primary antibodies used are listed in Supplemental Table S3.Blots were analyzed using the ImageQuant 800 imaging system (Amersham Biosciences, Amersham, UK) and quantification was performed using ImageJ 15 .Details can be found in the Supplemental Material.

Matrix metalloproteinase activity assays
Fluorescein conjugate gelatine assay -To measure the activity of collagenase and gelatinase, the supernatant of human coronary artery smooth muscle cells overexpressing ADAMTS7 or a mock plasmid was used with fluorescein conjugates of gelatine (DQ Gelatine from Pig Skin, ThermoFisher).Details can be found in the Supplemental Material.
Gel zymography -Supernatant of human coronary artery smooth muscle cells overexpressing ADAMTS7 or a mock plasmid was used.Details can be found in the Supplemental Material.
Colorimetric assay -Supernatant of HEK293 cells overexpressing ADAMTS7 (or mock plasmid) and TIMP1 were collected and subjected to concentration using 3K Amicon Ultra-15 filters (Sigma-Aldrich).Recombinant MMP-9 from the Matrix Metalloproteinase-9 Colorimetric Drug Discovery Kit (Enzo Life Sciences, New York, USA) was added to the supernatants.Assays were performed according to the manufacturer's recommendations.Absorbance was measured using an Infinite M200 PRO microplate reader (Tecan).

TIMP-1 degradation assays
Details can be found in the Supplemental Material.

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Europe PMC Funders Author Manuscripts In vitro degradation -HEK293 cells were transiently transfected with TIMP1 and ADAMTS7 constructs.Quantification was done using ImageJ as described above.
Flow cytometry -HEK293 cells were transiently transfected with TIMP1-GFP and ADAMTS7 constructs or a mock plasmid.The numbers and intensity of GFP-positive cells were measured by flow cytometry using a LSRFortessa (BD Biosciences, Franklin Lakes, USA) flow cytometer.
Fluorescence microscopy -HEK293 cells were transiently transfected with TIMP1-GFP and ADAMTS7 constructs.Analysis was performed by measuring the GFP intensity based on the cell count.To obtain cell counts we used a custom macro in ImageJ.

Analysis of ADAMTS7 expression in fibrous caps of human carotid artery plaques
Laser capture micro-dissection of advanced atherosclerotic carotid artery plaques was described previously 16 .In brief, carotid atherosclerotic lesions from the Munich Vascular Biobank 17 were subjected to microdissection of the fibrous cap and subsequent RNA isolation.RNA was isolated using the RNeasy Micro Kit (Qiagen) and cDNA was synthesized using the Taqman High-Capacity cDNA Transcription Kit (ThermoFisher).Primer assays for ADAMTS7 and RPLP0 as housekeeping gene (Hs00276223 and Hs00420895_gH, respectively; both ThermoFisher) were used to detect differences in gene expression between fibrous caps of stable and unstable plaques according to the histomorphologic AHA classification 18 , as described previously 16 .Baseline characteristics of included patients were described by Eken et al. 17 .ADAMTS7 mRNA levels were visualized and compared as 2 -ΔCt values.

Image selection and statistical analysis
For representative images, we chose the best illustrative and most representative images of the overall series of experiments with the best quality.Histological analyses were performed by an investigator who was blinded for the genotype.Randomization was not performed as all mice received the same treatment.All specimen which were technically usable for histological analysis were included.Normal distribution of data was assessed using the Kolmogorov-Smirnov test.Test results and subsequently used statistical tests are displayed in Suppl.Table S5.Data were analyzed using two-tailed Student's unpaired or paired t-test (for normally distributed data) or Mann-Whitney test (for non-normally distributed data), as appropriate and indicated in the respective figure legend (and Suppl.Table S5).
When comparing more than two groups, (RM) one-way repeated measures ANOVA test followed by an appropriate post-test for multiple comparisons was performed when data were normally distributed.To determine statistical outliers, the two-sided ROUT's test was used.No outliers were detected.Sample sizes/numbers of replicates are indicated in the figure legends and visualized in the figures (each symbol represents one animal/biological replicate) and data are displayed as mean and s.e.m.P-values below 0.05, in case of investigating more than two groups after adjustment for multiple testing, were regarded as statistically significant.Only within-test corrections were applied.Statistical analyses were performed using GraphPad Prism version 9 for macOS (GraphPad Software, La Jolla, CA, USA; RRID: SCR_002798).

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Results
Proteome-wide analysis of the matrisome of atherosclerotic plaques in Apoe-/-and Apoe-/-Adamts7-/-mice identifies Timp-1 as a novel target ADAMTS-7, an ECM protease 7 , was recently shown to be involved in mediating atherosclerotic plaque formation via its catalytic domain 19 .To unravel potential targets and substrates in atherosclerotic plaque formation, we performed a proteome-wide analysis of mouse aortae -focusing on ECM proteins -after feeding Apoe-/-and Apoe-/-Adamts7-/mice with a Western diet for 16 weeks.Using solubility profiling as described previously 12 , we detected more than 3,500 proteins of which 305 were associated with the ECM (Suppl.Table S6).The number of detected proteins in both genotypes was comparable (Suppl.Fig. S1).Expectedly, known targets of Adamts-7 in vascular remodeling, i.e., thrombospondin-1 (Thbs1) 8 and cartilage oligomeric matrix protein (Comp) 11 , were detected at higher protein levels in the ECM of Apoe-/-Adamts7-/-compared to Apoe-/-mice (Fig. 1B).Further proteins which were detected at higher levels in the absence of Adamts-7 include the tissue inhibitor of metalloproteinases (Timp) 1 (Fig. 1C).

Characterization of the interaction between ADAMTS-7 and TIMP-1
The finding that Timp-1 is detected at higher levels in Apoe-/-Adamts7-/-mice raised the question, whether it is a substrate of Adamts-7 and would directly interact with Adamts-7.
To explore this possibility, we first wished to confirm whether Adamts-7 and Timp-1 are co-localized in atherosclerotic plaques.In immunohistochemical stainings, we detected both Adamts-7 and Timp-1 in aortic root plaques of Apoe-/-mice after Western diet for 16 weeks.Immunofluorescence staining suggested co-localization of Adamts-7 and Timp-1 in the plaque region (Suppl.Fig. S2).Of note, the knockout of Adamts7 itself did not influence Timp1 mRNA levels (Suppl.Fig. S3).We next sought to investigate whether the proteins directly interact with each other.To that end, ADAMTS7-V5 and TIMP-1-HA constructs were ectopically expressed in HEK293 cells and protein lysates were subjected to co-immunoprecipitation.To determine the binding domain of TIMP-1 to ADAMTS-7, in addition to the full-length protein, we cloned the N-terminal region containing the catalytic domain and lacking the C-terminal thrombospondin repeats ( ΔTSPr ADAMTS7-V5) and the C-terminal part containing the disintegrin-like and the THBS1-like domains but lacking the catalytic domain ( Δcat ADAMTS7-V5) (Fig. 2A), and co-expressed these constructs with TIMP-1-HA.Following immunoprecipitation of TIMP-1-HA by an anti-HA antibody, ADAMTS7-V5 was revealed as co-precipitated protein by Western blot, indicating a direct protein-protein interaction (Fig. 2B).Importantly, the interaction was also seen when TIMP-1-HA was precipitated from the supernatant (Suppl.Fig. S4).The so far known targets of ADAMTS-7, i.e., TSP-1 and COMP, were shown to bind to the C-terminal region of ADAMTS-7 which contains the thrombospondin repeats 8,11 .In further Co-IP experiments, we found an interaction of TIMP-1-HA with the catalytic domain of ADAMTS-7 (Fig. 2C), whereas no interaction was detected with the C-terminal part containing the disintegrin-like and the THBS1-like domains (Fig. 2D).In summary, these data suggest that TIMP-1 belongs to the small group of ADAMTS-7 interacting proteins that bind to the catalytic domain rather than the C-terminal part of ADAMTS-7.

ADAMTS-7 leads to TIMP-1 degradation in vitro
We next sought to investigate whether TIMP-1 is degraded in the presence of ADAMTS-7.
We therefore performed an in vitro degradation assay using HEK293 cells transiently overexpressing full-length ADAMTS7-V5, the ADAMTS-7 construct lacking the N-terminal region and the catalytic domain ( Δcat ADAMTS7-V5), or mock transfection (empty vector).

Development of a high-throughput screening-usable protein-protein interaction assay
While the association of ADAMTS-7 with atherosclerosis has been clearly demonstrated in genetic studies in humans [4][5][6] and experimental studies in mice 8,9,19,22 , the molecular mechanism remained elusive.Using a proteome-wide analysis of atherosclerotic aorta in Apoe-/-and Apoe-/-Adamts7-/-mice after feeding a Western diet, we identified TIMP-1 as a putative candidate target and substrate.Inhibiting the protein-protein interaction between ADAMTS-7 and TIMP-1 might be a promising therapeutic approach to beneficially influence downstream effects of ADAMTS-7; assays that are suitable for high-throughput screening (HTS) to identify such inhibitors are, however, not available.Therefore, we sought to use time-resolved Förster resonance energy transfer (FRET) to quantify the interaction of ADAMTS-7 and TIMP-1.To that end, we cloned ADAMTS-7-FLAG and TIMP-1-HA constructs.After overexpression in HEK293 cells, protein lysates were incubated with anti-HA and anti-FLAG antibodies which were bound to the d2 and cryptate fluorophores, respectively.In this assay, we excited ADAMTS-7-FLAG-cryptate at 337 nm wavelength and measured the emission of cryptate at 620 nm and d2 secondary to FRET and 655 nm wavelength.The ratio between 665 nm and 620 nm (FRET ratio) was calculated as the primary readout.While there was no FRET signal detectable in the presence of ADAMTS-7-FLAG or TIMP-1-HA alone, the combination of ADAMTS-7-FLAG and TIMP-1-HA enabled us to detect a strong and reproducible FRET signal (ADAMTS-7-FLAG+TIMP-1-HA 5,484±302.9vs. mock+TIMP-1-HA 369.5±142.2,p=6.9•10 -10 , vs. ADAMTS-7-FLAG+mock 654.3±240.1 [665/620 ratio], p=5.5•10 -9 ; Fig. 6A).To verify that the FRET signal is specific for the interaction of ADAMTS-7 and TIMP-1 and to gain first insights into the feasibility as a screening assay for inhibitors, we performed a competition assay of ADAMTS-7-FLAG and TIMP-1-HA with high concentrations of untagged, recombinant TIMP-1.We found a dose-dependent decrease of the FRET signal in the presence of untagged TIMP-1 (untagged TIMP-1: 0 ng 4,954±197.5 vs. 400 ng 4,422±303.2vs. 800 ng 3,571±143.8[665/620 ratio], p for trend=2•10 -4 ; Fig. 6B), which is indicative of suitability to search for peptide or small molecule inhibitors.Further confirmation was done by co-expressing non-catalytic domain of ADAMTS-7 with TIMP-1.
As expected from the Co-IP results, no FRET signal indicating interaction was detected from overexpression of the non-catalytic domain of ADAMTS-7 with TIMP-1 (Suppl.Fig. S7).
Taken together, these results replicate the protein-protein interaction of ADAMTS-7 and TIMP-1 using a different, less artificial type of assay.As the binding of TIMP-1 to ADAMTS-7 is a prerequisite for scaffolding and degradation, this assay is suitable for screening against libraries of compounds to identify ADAMTS-7 inhibitors.

Discussion
ADAMTS-7 represents a novel CAD risk factor but its functional role in atherosclerosis and CAD remains incompletely understood.In this study, we performed a proteome-wide analysis of atherosclerotic aorta tissue from mice on a proatherogenic background which were fed a Western diet.Comparing Apoe-/-and Apoe-/-Adamts7-/-mice, we sought to identify proteomic differences in the presence and absence of Adamts-7.Specifically, bearing in mind the canonical role of ADAMTS-7 as an ECM protease, we focused on the ECM proteome using mass spectrometry analysis of tissue samples after solubility profiling as we described earlier 12 .In mice lacking Adamts-7, the known targets Comp and Thbs1 were detected at higher abundance which confirms previous observations from mechanistic studies in vascular remodeling 8,11 .In addition, we identified TIMP-1, an endogenous inhibitor of metalloproteinases 23 , to be detected at higher protein but not mRNA levels in aortic tissue from mice lacking Adamts-7.The family of TIMPs in humans consists of the four members TIMP-1, TIMP-2, TIMP3, and TIMP-4 (for an overview, see 23 ).The different members were shown to inhibit endogenous proteases with different affinities.While all TIMP2-4 were described to inhibit all or most endogenous MMPs, the spectrum of TIMP-1 is rather narrow; in particular, it seems to strongly interact with MMP-9 as it also binds pro-MMP-9.Importantly, considering other proteases particularly from the disintegrin family, TIMP-1 is only known to bind ADAM-10 while TIMP-3 in contrast inhibits a wide range of ADAM and ADAMTS proteases 24 .Since a biological interaction between ADAMTS-7 and TIMP-1 was not known before, we next aimed to verify that both proteins directly interact with each other.Using co-immunoprecipitation, we found that ADAMTS-7 and TIMP-1 directly bind to each other.Importantly, we were not able to demonstrate binding of TIMP-1 to the part of ADAMTS-7 containing the thrombospondin repeats.This is notable as the so far identified targets, including COMP and Thbs1 were shown to bind there 8,11 .In contrast, in this study TIMP-1 bound to the part of ADAMTS-7 containing the catalytic domain.This observation points to an important role of the catalytic domain in atherosclerosis which was recently also demonstrated in a study in which the deletion of only this part of ADAMTS-7 was sufficient to reduce atherosclerotic plaque formation 19 .Interestingly, a previous in vitro study showed that ADAMTS-7's protease function was not affected by TIMP-1, i.e., TIMP-1 itself does not inhibit ADAMTS-7 25 .Taken together, this renders an interaction between the catalytic domain of ADAMTS-7 and TIMP-1 likely to be involved in mediating risk of atherosclerosis.
Although ADAMTS-7 was described as a protease, we considered whether in addition to binding TIMP-1, ADAMTS-7 scaffolds or degrades it or is itself inhibited by TIMP-1.Of note, as described above TIMP-3 has been described to inhibit other ADAM and ADAMTS proteases, e.g., ADAM17 26 and ADAMTS-2 27 .In vitro degradation assays, however, revealed that in the presence of full-length ADAMTS-7 TIMP-1 is degraded while TIMP-1 levels remained stable in the presence of an ADAMTS-7 construct lacking the catalytic domain.Importantly, we observed similar findings using cell-based assays.The consistent in vitro findings along with the observation of increased Timp-1 levels in aorta samples from mice lacking Adamts-7 provide substantial evidence that i) TIMP-1 represents a novel target of ADAMTS-7 and ii) in contrast to TIMP-3, TIMP-1 does not mainly act as an inhibitor of this ADAMTS protease but rather as a substrate.
Previous studies clearly suggested a detrimental role of ADAMTS-7 in vascular biology 8,9,11,19,28 .The role of TIMPs and in particular TIMP-1, in contrast, remains controversial.In a mouse model of atherosclerosis using overexpression of Timp-1 or Timp-2, a study in a model of early atherosclerosis revealed a reduction of atherosclerotic plaques rather for Timp-2 than Timp-1 29 .In line, the same group reported that mice lacking Timp-1 did not develop larger plaques as compared to wild-type mice on a proatherogenic background 30 .This discrepancy might be due to the fact that TIMP-1 is a modulator of a broad range of biological processes.TIMP-1 can exert its effect both in an MMP-dependent and independent manner 31,32 .We here report a reduction of TIMP-1-mediated inhibition of MMP-9 due to scaffolding and degradation in the presence of ADAMTS-7 in vitro, given that MMP-9 is a bona fide interaction partner of TIMP-1 33 .MMP-9 itself is encoded by a CAD risk gene identified by genetic studies 21 and is one of the main proatherogenic proteases 34 .ECM remodeling caused by MMP-9 activity was shown to be involved in the transition from a stable to an unstable atherosclerotic lesion 20 .In this context, collagen content in the plaque is a readout of MMP-9 activity and we found less collagen content in Apoe-/-as compared to Apoe-/-Adamts7-/-mice.ADAMTS-7 was previously reported to be associated with a vulnerable carotid plaque phenotype 33 .Increased MMP-9 activity, as shown in our study, might contribute to this observation.In line, we found higher expression of ADAMTS7 in fibrous caps of unstable as compared to stable carotid plaques.In addition, it was shown that changes in ECM equilibrium due to MMP-9 activity induce cell-specific pro-inflammatory or pro-apoptotic activities 35 .As such, MMP-9 promotes proliferation Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts and migration of VSMCs 36 and proliferation and apoptosis of endothelial cells 23,37 , which contributes to remodeling of the tissue and atherosclerotic angiogenesis.The angiogenesisinducing potency of pro-MMP-9 is substantially decreased when encumbered by TIMP-1 38 .
In addition, TIMP-1 has MMP-9-independent cytokine-like activities 31,32 and can modulate a broad range of biological processes, e.g., cell growth, proliferation, apoptosis, migration, and angiogenesis, via binding to thus far unknown receptors and thereby inducing specific signaling cascades 39 .Thus, degradation of TIMP-1 by ADAMTS-7 could result in a plethora of further downstream processes regulated by interactions between TIMP-1 and MMP-9.
Taken together, we here provide evidence that the interaction of ADAMTS-7 and TIMP-1 contributes to the modulation of atherosclerotic plaques.As for other CAD risk factors identified by GWAS, there is hope that ADAMTS-7 could be used as a druggable target to prevent and treat atherosclerotic plaque formation.Assays which are able to quantify ADAMTS-7 activity are, however, so far lacking.As binding of TIMP-1 is a prerequisite of both scaffolding and degradation, we used FRET to quantify the protein-protein interaction between ADAMTS-7 and TIMP-1.Importantly, competition with untagged TIMP-1 was able to reduce the signal indicating a potential in screening efforts.This assay forms a sound foundation for it to be screened against relevant small molecule libraries to identify ADAMTS-7 inhibitors as well as modulators of the ADAMTS-7 and TIMP-1 protein-protein interaction.

Conclusions and limitations
We outlined a molecular mechanism involving the CAD risk gene ADAMTS-7 in atherosclerosis and plaque vulnerability (Graphical Abstract).TIMP-1, being degraded in the presence of ADAMTS-7´s catalytic site, represents a novel downstream target that partly explains the role of ADAMTS-7 in CAD.We are aware that ADAMTS-7 might have several further targets contributing to its effects in vascular biology.As such, the roles of COMP and TSP-1 remain to be further investigated.A further CAD risk gene identified by GWAS, sushi, von Willebrand factor type A, EGF and pentraxin domain-containing protein 1 (SVEP1) 40,41 was shown to be detectable at lower levels after overexpression of ADAMTS-7 before 8 .Current and future research efforts will therefore need to investigate the contribution of other downstream targets and their respective therapeutic potential.To that end, we established a FRET-based protein-protein interaction assay which may be used for high-throughput screening against libraries of small molecules to identify inhibitors that potentially prevent initiation or slow the progression of atherosclerosis.For large-scale screening efforts, limitations such as the preparation of cell lysates and incubation with antibodies need to be overcome.However, the assay can serve as a proof-of-concept for further development.Furthermore, due to the nature of the protein-protein interaction between ADAMTS-7 and TIMP-1, it is not possible to validate the mechanisms identified in vitro, i.e., reduced TIMP-1 availability, enhanced MMP-9 activity and subsequent collagen degradation, in a transgenic mouse model.Ideally, such models would include a murine TIMP-1 which cannot be degraded in the presence of ADAMTS-7.Finally, TIMP-1 as a putative target was identified in mouse aortas which by nature have differences to the human pathophysiology of CAD.The mouse, however, represents an established model in atherosclerosis research and due to the fact that we were able to confirm an interaction

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Europe PMC Funders Author Manuscripts between ADAMTS-7 and TIMP-1 using the human proteins renders conservation of this interaction in humans likely.A. Volcano plot displaying proteins that were more abundant in Apoe-/-Adamts7-/-mice (right) as compared to Apoe-/-mice (left).Red and blue dots represent proteins which were significantly different between the genotypes after correction for multiple testing.B.
The known ADAMTS-7 targets Comp and thrombospondin 1 (Thbs1) were detected at numerically lower levels in aortic tissue of Apoe-/-as compared to Apoe-/-Adamts7-/-mice.C. As putative novel targets of ADAMTS-7, we observed higher levels for the endogenous tissue inhibitors of MMPs (Timp) Timp-2 and, in particular, Timp-1 (Timp2 was detected

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Europe PMC Funders Author Manuscripts ADAMTS7 mRNA was detected at higher levels in caps of unstable (n=10) as compared to caps of stable plaques (n=10).Mann-Whitney test.